Method of producing surfactin with the use of mutant of bacillus subtilis

ABSTRACT

The present invention relates to a stable mutant of Bacillus subtilis which can produce surfactin with high yields, a method of producing surfactin with the use of the strain and the use of the surfactin obtained for pharmaceutical, energy and environmental problems.

DESCRIPTION

The present invention relates to a stable mutant of Bacillus subtiliswhich can produce surfactin with high yields, a method of producingsurfactin with the use of the strain, and the use of the surfactinproduced for pharmaceutical, energy and environmental problems.

In recent years interest in biosurfactants of microbial origin for useas agents in the assisted recovery of hydrocarbons, the stabilisation ofemulsions and, more generally, in the energy and environmental fieldshas increased considerably since these products are biodegradable andhence potentially less toxic than the synthetic compounds currentlyused.

A biosurfactant of particular interest, which is produced by Bacillussubtilis, is surfactin.

This compound, which was characterised by Kakinuma et al. [Agric. Biol.Chem., 33: 971-972 (1969); Agric. Biol. Chem., 33: 1523-1524 (1969);Agric. Biol. Chem., 33 973-976 (1969)], is a cyclic lipopeptide formedby a heptapeptide and a lipid portion constituted by a mixture ofbeta-hydroxy-fatty acids with chains having between 13 and 15 carbonatoms and has the following structure: ##STR1##

Surfactin has the property of inhibiting the formation of blood clotsand 3', 5' monophosphate diesterase and of lysing erythrocytes,spheroplasts and bacterial protoplasts.

Moreover, surfactin inhibits the fibrinogen-thrombin reaction, thusslowing the formation of fibrin; this property makes the substancesuitable as an active element for the preparation of compositions usefulas anticoagulants in the prophylaxis of thrombosis and for preventingdiseases such as myocardial infarction, pulmonary embolism, etc., ingeneral.

Surfactin shows anti-cholesterase activity, since it lowers the levelsof cholesterol in the plasma and in the liver, as well as fungicidal andantibiotic activity.

The lipopeptide performs its bacteriostatic functions, for example, onthe growth of mycobacteria, even at low concentrations (5-10 ppm).

Moreover, surfactin is a powerful surface-active agent and in factreduces the surface tension of water from 72 mN/M to 27 mN/M at aconcentration of 0.005%.

Its multiple activity means that surfactin is of particular interest,since it can be used widely in the pharmaceutical, energy andenvironmental fields.

Arima, K et al. (U.S. No. 3,687,926 and Biochem, Bioph. Res. Commun.,31: 488-494 (1968)) describe a method of perparing surfactin having thefollowing structure: ##STR2## characterised by the use of the B.subtilisstrain ATCC 21331 or ATCC 21332.

However, this method has disadvantages resulting from the low yields ofsurfactin (0.05-0.1 g/litre of the crude product and 0.04-0.05 g/1 ofthe purified product).

Consequently, this method is not very acceptable economically from acommercial point of view.

Methods of improving surfactin yields have therefore been proposed inthe art and these are based, essentially, on the use of particularculture media or mutants of the B.subtilis strain ATCC 21332 orparticular technical solutions.

Thus, for example, Cooper D.G. et al., [(1981), Appl. Environ.Microbiol., 42: 408-412)]describe a method of producing surfactin by theculture of B.subtilis ATCC 21332 based, amongst other things, on thecontinuous removal of the foam which is produced during the fermentationand which contains 90-99% of the surfactin produced.

The object of removing the foam is to prevent the inhibiting effectwhich high concentrations of surfactin have on bacterial growth.

Under these conditions, however, a surfactin yield of 0.7-0.8 g/litre isachieved.

Moreover, the continuous removal of the foam may reduce the workingvolume of the fermentation medium which is discharged with the foam.

Sheppard J. and Mulligan C., (1987), (Appl. Microbiol. Biotechnol., 27:110-116) describe a method by which a yield of 0.16 g/litre is obtainedby the growth of B.subtilis cells in a medium supplemented with ahydrolysed peat protein.

Moreover, Mulligan, C. et al [Appl. Microbiol. Biotech., 31: 486-489(1989)]describe a method for improving the yield of surfactincharacterised by the use of a mutant of the B.subtilis wild-type strainATCC 21332.

The authors report a surfactin yield of 0.562 g/litre after 40 hours (p.488, Table 1), that is, about 3.4 times greater than that obtained bythe growth of the wild-type strain under the same conditions.

In spite of the large amount of work carried out, therefore, no proposalhas been found cheap enough to enable it to be developed on anindustrial scale, mainly because of the low productivity of themicro-organisms available hitherto.

It has now been found that the problems of the prior art can be overcomeby a new mutant of B.subtilis which can produce surfactin with highyields.

Samples of this mutant strain were deposited at the American TypeCulture Collection on Apr. 23, 1990 and received the registration numberATCC 55033.

A subject of the present invention is therefore the B.subtilis strainATCC 55033.

A further subject of the present invention is a method of producingsurfactin with high yields including the use of the B.subtilis strainATCC 55033.

Another subject of the present invention is the use of the surfactinproduced in the pharmaceutical, energy and environmental fields.

Further subjects of the present invention will become clear from thefollowing description and examples.

In particular, the B.subtilis strain ATCC 55033 according to the presentinvention is characterised by good genetic stability (the ability toretain the mutation acquired permanently) and good resistance to highconcentrations of surfactin.

This strain was produced by the mutation of the B.subtilis wild-typestrain ATCC 2i332, which is commonly available to the public. For thispurpose, it is possible to use conventional methods consisting ofexposing cells of the wild-type strain to the action of chemical orphysical mutagenic agents, selecting the strains in which the surfactinyield is altered and, finally, isolating those colonies in whichproductivity is increased.

Chemical mutagenic agents may be selected, for example, from diethylsulphate, NMU (nitrosomethyl urethane), NMG(N-methyl-N'-nitro-N-nitrosoguanidine), and physical agents may beselected from X-rays, UV-rays (ultra-violet) and gamma rays in mutagenicdoses.

According to one embodiment of the present invention, the B.subtiliswild-type strain ATCC 21332 was mutagenised with the use of NMG inconcentrations such as to induce mutations in the genomes of themicro-organisms.

The mutants which could produce surfactin with high yields (theoverproducer mutants) were then selected by analysing the sizes of thehaemolysis haloes which appeared around the B.subtilis colonies grown ona culture medium such as, for example, TBAB (DIFCO) to which blood hadbeen added.

In fact, it is known that the diameter of the haemolysis halo isproportional to the quantity of surfactin produced by the cells ofB.subtilis (Mulligan, C. and Cooper, D. (1984), J. Ferment. Technol.,62: 158-179).

The overproductivity of the B.subtilis mutant ATCC 55033 thus isolatedwas then confirmed by the fermentation of the strain in question in aflask with the use of the wild-type strain as a control.

B.subtilis ATCC 55033 seems to be particularly suitable for producingsurfactin with high yields by a fermentation method.

A method according to the invention may consist, for example, ofpreparing an inoculum of the mutagenised strain under aerobic conditionsin an aqueous medium containing assimilable sources of carbon andnitrogen.

The medium is kept under agitation at a temperature between 25° and 40°C., preferably from 30° to 37° C. for a period shorter than 20 hours,preferably from 6 to 10 hours.

In fact, it has been found that an "old" inoculum (20 hours) causes foamproduction which is difficult to control to the point that after 5 hoursthe fermentation has to be stopped because of the extensive loss of theculture medium which is discharged with the foam.

"Young" inocula (6-10 hours) on the other hand enable the foam to formgradually with a limited loss of the culture medium.

A percentage of the inoculum of between 5% and 10% (volume/volume) ofthe working volume is then added to the fermentation medium whichcontains assimilable sources of carbon and nitrogen as well as variouscations, anions and possibly vitamins such as biotin or thyamine and anaminoacid suitable for encouraging cell growth and the production ofsurfactin, selected from L-Valine, L-Leucine, D-Leucine andL-Isoleucine.

The initial cell density of the fermentation is generally equivalent toan 0.D.₆₀₀ of between 0.025 and 0.040.

Assimilable sources of carbon include carbohydrates such as glucose,hydrolysed starches, molasses, sucrose or other conventional carbonsources.

Examples of nitrogen sources may be selected, for example, from mineralammonium salts such as ammonium nitrate, ammonium sulphate, ammoniumchloride or ammonium carbonate, urea, or products containing organic orinorganic nitrogen such as peptone, yeast extract or meat extract.

The following cations and anions are also suitable for the purposes ofthe present invention: potassium, sodium, magnesium, iron, calcium, acidphosphates, sulphates, chlorides, manganese and nitrates.

According to the present invention a fermentation medium having thecomposition given in Example 2 is preferred.

The fermentation is carried out in a vessel (a fermenter or bioreactor)with stirring and intensive aeration, at a temperature usually between25° and 40° C., preferably between 30° and 37° C., with the continuousremoval of the foam formed which contains more than 98% of the surfactinproduced.

Quantities of sterile compressed air which may vary from 0.2 to 1.0vol/vol/minute are diffused into the fermentation medium.

The pH of the fermentation medium is kept between 6.0 and 7.2 andpreferably between 6.7 and 6.9.

The pH may be adjusted, for example, by the addition of a basic aqueoussolution such as an aqueous solution of ammonia, potassium hydroxide,sodium hydroxide, sodium carbonate or potassium carbonate.

It is preferable, however, to keep the pH to the desired value with theuse of 10N sodium hydroxide (NaOH).

The stirring speed, which is one of the reasons for the production ofthe foam, is generally selected so as to allow high bacterial growthwithout having dramatic effects on the formation of the foam.

The initial stirring speed is typically between 150 and 400 rpm,preferably between 200 and 300 rpm.

Finally, the working volume of the fermentation which appears to be acritical element as regards the formation of the foam, is selected so asto limit the production of foam and hence the loss of the cellularbiomass which is discharged with the foam produced.

The removal of the foam formed during the fermentation process reducesthe working volume and results in the loss of the cells containedtherein.

There are two possible approaches to overcome this problem. In one case,fresh sterile culture medium may be added to the bioreactor continuously(continuous fermentation) and, in the second case, the culture broth.(containing the bacterial cells) which is discharged with the foam maybe recycled into the bioreactor (the recycling of the culture medium).

According to a preferred embodiment of the method of the presentinvention, the culture medium which is eliminated with the foam removedis recycled to the bioreactor with the use, for example, of an automaticsystem as shown in FIG. 8.

Such a fermentation could present problems due to the fact that theconcentration of the surfactin in the recycled medium could have anadverse effect on the further production of surfactin in the bioreactorbecause of the inhibiting effect of the substance on bacterial growth(Cooper et al.).

Surprisingly, however, the results obtained have shown an increase inthe biomass and constant surfactin values in the bioreactor.

This indicates that the supply of a concentrated solution of surfactinfrom the collection container to the bioreactor has a limited effect oncell growth and that the surfactin has no inhibitory effect on its ownsynthesis by the mutant B.subtilis strain ATCC 55033.

These results suggest that, unlike the B.subtilis wild-type strain ATCC21332, the mutant of the present invention may be more resistant to highconcentrations of surfactin.

According to a further embodiment of the method of the presentinvention, the foam and the culture medium removed from the bioreactormay be treated by an ultrafiltration system assembled as shown in FIG.12. The culture medium is then recycled to the bioreactor without thesurfactin.

When the fermentation is carried out under the preferred conditions, aconcentration of crude surfactin of from 2.0 to 4.0 g/litre is obtainedwithin a period of from 40 to 90 hours.

At the end of the fermentation process, the surfactin is recovered fromthe foam and from the a cellular or cellular supernatant liquid andpurified.

Conventional methods may be used for this purpose, such as, for example,precipitation by an inorganic acid such as sulphuric or hydrochloricacid or by a compound of a bivalent metal such as calcium, or bysaturation with ammonium sulphate.

This treatment enables the selective precipitation of the surfactin andof some lipopeptides and lipoproteins produced by B.subtilis.

This precipitation step may be preceded by the treatment of the acellular supernatant liquid by an ultrafiltration system in order toremove coarser impurities and concentrate the working volume to bepurified.

The precipitate containing the surfactin is then purified with the useof one of the known techniques such as, for example, extraction withorganic solvents such as chloroform or methylene chloride or salineprecipitation by CaCl₂ or NaCl.

The method of the present invention produces quantities of from 1.2 to2.0 g/litre of purified surfactin (99%).

The chemical-physical characterisation of the surfactin produced by themethod of the present invention may be carried out by conventionalmethods with the use of chromatographic, spectroscopic or spectrometrictechniques.

The results obtained by mass spectrometry (FAB), infrared spectrometry(IR), nuclear magnetic resonance (NMR) and high-pressure liquidchromatography (HPLC) confirmed the data in the literature.

Moreover, the presence of fatty-acid molecules which differ not only inthe length of their carbon chains (C₁₃ -C₁₅) but also in theirstructures which may be normal, iso or anteiso was confirmed.

The characterisation of the surface-active and aggregative properties ofthe surfactin produced by the method of the present invention confirmedthe data in the literature.

The surfactin obtained by the method according to the present inventionis therefore particularly suitable as a surface-active agent, as astabilising agent for therapeutic compounds such as medication for thetreatment of thromboses, embolisms and inflammation, and in the energyand environmental fields.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

A photograph of a plate of TBAB (DIFCO) medium to which blood has beenadded, showing the haemolysis haloes of the B.subtilis wild-type strainATCC 21332 and of the B.subtilis overproducer mutant ATCC 55033.

FIG. 2

A calibration curve in which the weight (g/1) of the solid biomass isshown on the ordinate and the absorbance at 600 nm is given on theabscissa.

FIG. 3

A curve correlating the diameter (cm) of the haemolysis halo produced onTBAB medium to which blood has been added (on the ordinate) with thequantity of surfactin (mg/ml) added to the medium (on the abscissa).

FIG. 4

A growth curve for the B.subtilis mutant ATCC 55033 showing time as afunction of the absorbance measured at a wavelength of 600 nm andcompared with the growth curve of the wild-type strain.

The time is expressed in hours on the abscissa and the absorbance(0.D.₆₀₀) on the ordinate.

FIG. 5

A graph showing the cell growth and surfactin production detected duringthe fermentation of the B.subtilis mutant ATCC 55033 in a flask.

The time is shown in hours on the abscissa; the absorbance value of thecell culture at a wavelength of 600 nm is shown (on a logarithmic scale)on the ordinate on the left-hand side, and the quantity (g/1) ofsurfactin produced by B.subtilis ATCC 55033 is shown on the right-handside.

FIG. 6

A graph showing the cell growth detected during the fermentation of theB.subtilis mutant ATCC 55033 in a flask in the presence of theaminoacids Val, Leu and Ile (*) and of Ile (o).

The time is shown in hours on the abscissa; the absorbance of the cellculture at a wavelength of 600 nm is shown (on a logarithmic scale) onthe ordinate on the left-hand side.

FIG. 7

A graph showing the surfactin production during the fermentation of theB.subtilis mutant ATCC 55033 in a flask in the presence of theaminoacids Val +Leu +Ile (*) and Ile (o).

The time is shown in hours on the abscissa; the quantity (g/1) ofsurfactin produced is given on the ordinate on the right-hand side.

FIG. 8

A diagram showing a 2-litre fermenter with recycling in which:

1) is the fermenter, 2) is an air-inlet line, 3) is a pump, 4) is aused-air line, 5) is a foam-collection container and 6) is the line forrecycling the culture medium.

FIG. 9

This drawing shows the rate of growth of the B.subtilis strain ATCC55033 achieved in a 2-litre fermenter with the use of inocula ofdifferent ages.

The time is shown in hours on the abscissa; the growth rate is shown onthe ordinate.

FIGS. 10 and 11

Graphs showing the cell growth and surfactin production detected duringthe fermentation of the B.subtilis mutant ATCC 55033 "with recycling".

The time is shown in hours on the abscissa; the absorbance value of thecell culture at a wavelength of 600 nm is shown (on a logarithmic scale)on the ordinate on the left-hand side, and the corresponding quantitydetected by the measurement of the areas of the three mainchromatographic peaks in which the presence of surfactin was shown isindicated on the right-hand side.

FIG. 12

A diagram showing the production of surfactin with the continuousultrafiltration of the product, in which: 1) is the fermenter, 2) is anair-inlet line, 3) is a pump, 4) is a used-air line, 5) is afoam-collection container, 6) is an ultrafiltration cartridge and 7) isa container for the recycled culture medium.

FIG. 13

A) shows comparative curves of the growth of the B.subtilis mutant ATCC55033 during fermentation with recycling and with ultrafiltration. Thetime is shown in hours on the abscissa; the absorbance value of the cellculture at a wavelength of 600 nm is given (on a logarithmic scale) onthe ordinate.

B) compares the quantities of surfactin present in the bioreactor in afermentation test with ultrafiltration and in a fermentation test withrecycling.

The time is shown in hours on the abscissa; the quantity of surfactin(g/1) is given on the ordinate.

FIG. 14

A calibration curve correlating the quantity of surfactin expressed ing/1 (abscissa) with the resulting value of the sum of the areas of sixpeaks produced by HPLC analysis (ordinate).

FIG. 15

A chromatographic profile of a 25 μg sample of purified surfactin. Thethree main peaks are constituted by surfactin bound to fatty acids withlengths varying between 13 and 15 carbon atoms.

FIG. 16

The chromatographic profiles of this drawing are the result of thedirect analysis of 50 μl of the acellular supernatant liquid withdrawnduring fermentation. The profiles relate to samples taken 3, 4, 27 and30 hours after the start of the experiment. The presence of the productis shown clearly by the appearance and subsequent development of thechromatographic peaks relating to the various components of surfactin(C13-C14-C15)

FIG. 17

The characterisation of a sample of purified surfactin by the FAB (FastAtom Bombardment) technique. The main peaks shown relate to productshaving molecular weights of 1008, 1022 and 1036 respectively, andcorresponding to surfactin fractions bound to beta-fatty acids withchains of various lengths.

FIG. 18

An IR (infrared ray) spectrum of a sample of purified surfactin. Theinterpretation of the spectrum is given in Example 6C.

The present invention will be described further by the followingexamples.

EXAMPLE 1 Preparation of the B.subtilis mutant which is an overproducerof surfactin

A preculture of the B.subtilis wild-type strain ATCC 21332 (availablefrom the American Type Culture Collection) was grown at 37° C. for onenight on Schaeffer sporulation medium having the following composition:

    ______________________________________                                        Nutrient broth (DIFCO)                                                                             8.0        g/l                                           KCl                  1.0        g/l                                           MgSO.sub.4           1.25 × 10.sup.-1                                                                   g/l                                           Agar (DIFCO)         16.0       g/l                                           MnCl.sub.2.4H.sub.2 O                                                                              1.98 × 10.sup.-3                                                                   g/l                                           FeSO.sub.4.7H.sub.2 O                                                                              2.78 × 10.sup.-4                                                                   g/l                                           Na.sub.2 SO.sub.4    1.42 × 10.sup.-1                                                                   g/l                                           H.sub.2 O            1          liter                                         pH                   7.0                                                      ______________________________________                                    

A loop of the preculture was then used to inoculate 10 ml of DIFCO VYmedium (Veal Infusion Broth 25 g/1 and yeast extract 5 g/1) and grown at37° C. for 16 hours.

A portion (100 μl) of the culture was transferred into a 100 ml flaskcontaining 10 ml of fresh VY medium and grown with gentle stirring (200revolutions per minute, rpm) at 37° C. until an optical density (0.D.)of 0.7 determined at 600 nm by a Beckman spectrophotometer (mod. DU70)was achieved.

The bacterial cells were then separated from the supernatant liquid bycentrifuging at 5,000 rpm for 10 minutes with the use of a Mod. SS. 34rotor on a Sorvall supercentrifuge, washed with 5 ml of TM buffer(Tris-HCl 0.05M, maleic acid 0.05M, (NH₄)₂ SO₄ 1 g/litre, MgSO₄.7H₂ O,0.1 g/litre, Ca(NO₃)₂ 5 mg/litre, FeSO₄.7H₂ O0.25 mg/litre) brought topH 6.0 with 5N NaOH, separated again by centrifuging in the mannerdescribed above, and resuspended in 5 ml of TM buffer.

5 ml of a solution of hydrated N-methyl-N'-nitro-N-nitrosoguanidine (1:1with H₂ O) (Fluka) in TM buffer (0.3 mg/ml) was then added to thecellular suspension and then incubated with stirring at 37° C.

After 30 minutes the suspension was centrifuged again and the cellsrecovered were washed with 5 ml of TM buffer and then resuspended in 50ml of fresh VY medium.

Portions (1 ml) of the suspension were grown with gentle stirring at 37°C. for one night and, after the addition of 0.2 ml of sterile glycerol,were frozen at -80° C.

The B.subtilis mutants were then selected by the spreading of serialdilutions (about 2×10² cells/plate) of the portions on TBAB mediumplates (Tryptose Blood Agar Base (DIFCO) 33 g/litre) to which 5% ofdefibrinated ram's blood (SCLAVO S.p.A.,) filtered through sterile gauzehad been added, after sterilisation at 120° C. for 20 minutes.

After thermostatically-controlled incubation at 37° C. for 16-24 hours,the diameter of the haemolysis haloes which appeared around thebacterial colonies were determined (FIG. 1).

In fact, it is known that the size of the haemolysis halo isproportional to the quantity of surfactin produced by the B.subtiliscells (Mulligan, C. and Cooper, D. (1984), J. Ferment. Technol., 62: 158-179).

One of the surfactin-overproducer colonies, designated B.subtilis SMS274, was deposited as ATCC 55033.

EXAMPLE 2 Production of surfactin in a flask

A colony of B.subtilis SMS 274 was used to inoculate 10 ml of VY mediumand grown at 200 rpm, at 37° C. for 16 hours.

100 μl of this preculture were then transferred into a flask with acapacity of 2 litres containing 1 litre of minimum medium having thefollowing composition:

    ______________________________________                                        Glucose            40.00      g/l                                             NH.sub.4 Cl        4.00       g/l                                             KH.sub.2 PO.sub.4  4.00       g/l                                             NaHPO.sub.4        5.64       g/l                                             MgSO.sub.4.7H.sub.2 O                                                                            0.20       g/l                                             CaCl.sub.2.2H.sub.2 O                                                                            1.00 × 10.sup.-3                                                                   g/l                                             FeSO.sub.4.7H.sub.2 O                                                                            20.00 × 10.sup.-3                                                                  g/l                                             MnCl.sub.2.4H.sub.2 O                                                                            1.98 × 10.sup.-4                                                                   g/l                                             EDTA               1.50 × 10.sup.-3                                                                   g/l                                             pH                 7.0                                                        ______________________________________                                    

The culture was carried out with stirring (250 rpm) at 37° C. for 24 and48 hours in a New Brunswick thermostatically-controlled incubator.

In all the experiments the B.subtilis strain ATCC 21332 grown under thesame conditions was used as a control.

Cell growth (biomass) was monitored by the determination of the opticaldensity of the culture broth at 600 nm (DU 70 spectrophotometer, BeckmanInstruments, Inc., USA) with the use of dishes with optical paths of 1cm (Bio-Rad Laboratores, USA). The O.D. values were then converted tog/1 by means of a standard curve produced with the use of variousdilutions (of known weights) of the solid biomass of the micro-organism(FIG. 2).

The surfactin was determined by the deposition of portions of the acellular supernatant liquid produced after the centrifuging of 2 ml ofculture broth (Biofuge A, Heraeus Sepatech, FRG) at 7,000 rpm for 5minutes at 4° C. on TBAB medium plates to which blood had been added andthe determination of the sizes of the haloes formed

The supernatant liquid was diluted by 1:10,000 with distilled water.

The degrees of haemolysis of the blood cells (the sizes of the haloes)were used as indications of the surfactin productivities of themicro-organisms.

Estimates of the yields were made by means of a standard curvecorrelating the sizes of the haemolysis haloes with known concentrationsof surfactin (FIG. 3).

FIG. 4 shows the growth curves of the mutant SMS 274 and of thewild-type strain ATCC 21332 obtained by the determination of the O.D.₆₀₀values over a period of time.

It can be seen from an analysis of this drawing that the growth of theB.subtilis mutant SMS 274 is wholly comparable to that of the wild-typestrain.

FIG. 5 shows the surfactin growth and production curves for the mutantSMS 274 and for the wild-type strain.

It can be seen from this drawing that determinable levels of surfactinare present 10 hours after the start of the fermentation, that is, atthe start of the truly logarithmic stage. The amount of surfactinpresent in the fermentation medium increases linearly, in comparisonwith the stationary growth phase, at an accumulation rate (ΔP/Δt) ofabout 0.32 g/1h. A surfactin value of 3.5 g/1 is reached during thestationary phase. In this test only the quantity of surfactin present inthe culture medium, which is approximately comparable to the totalsurfactin yield, was determined.

EXAMPLE 3 Investigation of the effect of aminoacids on the growth andproduction of surfactin by B.subtilis SMS 274

A series of tests was carried out to check the effects of aminoacids onthe growth and production of surfactin by the mutant SMS 274.

The tests were carried out as in Example 2 in 2-litre flasks eachcontaining 1 litre of minimum medium supplemented with 5 mg/1 of each ofthe following aminoacids: L-Leucine (Leu), L-Valine (Val), L-Isoleucine(Ile) and D-Leucine.

The results showed that the presence of the three aminoacids L-Leu,L-Val and L-Ile in the fermentation medium induced an increase in thebiomass (FIG. 6).

During the first 20 hours, the surfactin production was similar to thatobtained in the control (the same strain grown in a medium withoutadditional aminoacids), however, the surfactin content in the mediumincreased during the stationary phase and at the end of the fermentationthe quantity of surfactin was about 30% greater than that found in thecontrol (FIG. 7) and Table 1.

When only Ile was added to the fermentation medium, it was observed thatcell growth was inhibited and there was a delay of 1 hour in theformation of surfactin. 60 hours after the start of the fermentation,however, there was an increase of about 20% in the surfactin produced incomparison with the control and the surfactin to cell ratio was 1.19,indicating an increase in the cellular production of surfactin.

                  TABLE 1                                                         ______________________________________                                                    srf.sub.60                                                                              OD.sub.60  srf.sub.60 /biomass                                      g/l       600 nm     g/l per g/l                                  Aminoacids  (+/-10%)  (+/-5%)    (+/-10%)                                     ______________________________________                                        Val + Ile + Leu                                                                           4.5       18.8       0.75                                         Ile         4.2       11.0       1.19                                         D-Leu       3.6       16.0       0.74                                         control     3.5       15.0       0.74                                         ______________________________________                                         Note: the surfactin srf was determined by HPLC analysis after 60 hours.  

EXAMPLE 4 Production of surfactin in a fermenter with recycling

The object of the test was to check the effects on the surfactin yieldof parameters such as the age of the inoculum, the stirring and theworking volume.

A slant preculture of the strain B.subtilis SMS 274 was used toinoculate a 100 ml flask containing 10 ml of VY medium and incubatedwith gentle stirring at 37° C. for 16 hours.

500 ml flasks each containing 100 ml of minimum medium were then eachinoculated with 1 ml of the preinoculum and some were kept at 37° C.with stirring at 350 rpm for 6 hours ("young" inocula) and others for 20hours ("old" inocula).

The cultures (100 ml) were then used to inoculate a fermenter with acapacity of 2 litres controlled by an electronic control unit(Instrumentation Laboratories) for monitoring and automaticallycorrecting the temperature, the pH, the dissolved oxygen and thestirring speed.

The inocula were added to 1 litre of minimum medium sterilised directlyin the fermenter, producing final working volumes of from 1.1 to 1.6litres.

The initial optical density of the culture (at the time t₀), determinedat 600 nm with the use of a Beckman spectrophotometer (mod. DU 70) was0.035 on average.

The initial fermentation conditions were:

    ______________________________________                                        temperature      37° C.                                                aeration         0.6-0.7 vol/vol/minute                                       stirring         200-700 rpm                                                  pH               6.8-6.9                                                      ______________________________________                                    

These parameters were kept constant automatically throughout thefermentation period and, in particular, the pH was kept at about 6.8 bythe addition of 10N NaOH.

The fermentation period was between 10 and 90 hours.

During the fermentation, the initial stirring speed of 200-220 wasbrought to about 400 rpm to keep the p₀₂ >15%.

The foam produced during the fermentation was removed continuously withthe use of an exhaust-air outlet line of the fermenter (FIG. 8).

About 20 hours after the start of the fermentation, that is, when anappreciable amount of surfactin and hence of foam had been produced, asystem for recycling the culture medium discharged from the bioreactorwith the foam was activated by means of a pump in order to keep theworking volume constant.

During the fermentation, the cell growth was monitored by thedetermination of the optical density at 600 nm and the surfactin in thea cellular supernatant liquid was determined by HPLC analysis asdescribed in Example 8.

The data obtained showed that:

the best results were obtained with initial stirring at from 200-220rpm; in fact faster stirring (500-700) caused foam production which wasdifficult to control to the extent that 5 hours after the start of thefermentation the experiment had to be stopped because of the excessiveloss of the culture broth.

unlike a "young" inoculum (6 hours), an "old" inoculum (20 hours) causedrapid and uncontrolled foam production only 5 hours after the start ofthe fermentation (FIG. 9).

a working volume ≦1.6 litres (from 1.1 to 1.41) is preferable.

Under the preferred conditions, cell growths equivalent to 12 and 15units of absorbance (0.D. 600), corresponding to about 4 g/1 of solidbacterial biomass (for a final O.D.₆₀₀ of 15) were observed after 47hours (FIG. 10) and 80 hours (FIG. 11), respectively. The surfactin waspurified from the acellular supernatant liquid and from the foamresulting from the fermentation by the method described in point b) ofExample 7.

The results in terms of the yield of the purified product obtained after47 and 80 hours were 1.20 g/1 and 2.0 g/1, respectively.

EXAMPLE 5 Fermentation with the continuous ultrafiltration of thesurfactin

The system included an on-line ultrafiltration system connected to thefermenter (FIG. 12).

The system proposed provided for the continuous removal of the surfactinproduced during the fermentation by the treatment of the foam and someof the fermentation broth (that which was discharged as a result of thepressure created in the fermenter) in ultrafiltration equipment (Amiconmod. CH2A) constituted by a collection container, a peristaltic pump anda hollow-fibre ultrafiltration cartridge (Amicon mod. HlP30-43) with anominal cut-off of 30,000 daltons.

As shown in FIG. 12, the liquid from the fermenter passed through thecartridge (6 ) and most of the surfactin (more than 95%) was retained inthe ultrafiltration system in the form of aggregates. A pump system (3)returned the culture medium to the fermenter without the product inquestion.

During the fermentation, cell growth was monitored (by the determinationof the optical density at 600 nm) and the production of surfactin wasmonitored (by the chromatographic technique described in Example 8,point A). Table 2 shows comparative data from a fermentation test withultrafiltration and from a fermentation test with a recycling systemassembled as in Example 4.

                  TABLE 2                                                         ______________________________________                                                      Surfactin                                                       Sections        Ultrafiltration                                                                            Recycling                                        ______________________________________                                        Fermenter       0.046 g       3.157 g                                         Foam            --            0.706 g                                         Filtered liquid 0.082 g      --                                               Retained liquid 3.002 g      --                                                               3.002 g       3.863 g                                         Final optical   7.2 units    15.5 units                                       density O.D..sub.600                                                          Surfactin/biomass                                                                             1.36*         0.78*                                           ______________________________________                                         where:                                                                        * = grams of surfactin per gram of biomass per liter calculated according     to the following formula:                                                     ##STR3##                                                                      65 hours after the start of the fermentation, the cell growth was             equivalent to 7.2 absorbance units (O.D..sub.600) corresponding to about      2.3 g/liter by weight of solid bacterial biomass.   system was then           purified from the acellular medium by the method described in point b) of     Example 7.

The result in terms of the yield of purified surfactin was 1.88 g/litre.

FIG. 13 shows the data for cell growth (A) and the efficiency of theremoval of the surfactin (B) compared with those achieved byfermentation with recycling alone.

EXAMPLE 6 Production of surfactin in a 20-litre fermenter with recycling

A SETRIC G.A. fermenter mod. SET.20 with a capacity of 20 litres wasused and contained 11 litres of minimum medium having the compositiongiven in Example 2.

A 6-hour preculture (1 litre) of B.subtilis SMS 274 produced asdescribed in Example 4, was transferred into the fermenter (a 10%inoculum). The final working volume was thus 12 litres.

In this experiment a system for continuously removing the foam producedand recycling the fermentation medium was also used.

The fermentation was carried out at 37° C. with aeration at 0.5vol/vol/minute, initial stirring at 200-220 rpm and a pH of about 6.8,kept at that value by 10N NaOH solution.

The other parameters were kept constant automatically throughout theduration of the experiment.

17-18 hours after the start of the fermentation, the foam generated inthe surface layer in the reactor was collected in the collectioncontainer together with some of the fermentation medium and, after 28hours, the pump for recycling the fermentation medium was activated tore-establish the working volume in the bioreactor.

During the stationary phase, the cell growth reached an O.D.₆₀₀ of 6.6with an increase 5-6 times that obtained in a 2-litre fermenter.

At the end of the fermentation 21 g (1.8-1.9 g/1) of purified surfactinwere obtained.

EXAMPLE 7 Isolation and purification of the surfactin

After the bacterial cells had been removed by centrifuging at 5,000 rpmfor 10 minutes in a Sorvall centrifuge with the use of a mod. GS.3rotor, the surfactin may be recovered from the accellular supernatantliquid and from the foam from the fermentation process by twoalternative methods:

a) Concentration by ultrafiltration followed by extraction as in point(b)

This treatment has the objects of (1) reducing the volume to be treatedin the subsequent purification step (b) and (2) removing from thesupernatant liquid compounds with low molecular weights which might beprecipitated together with the surfactin during the subsequentpurification step (b).

Portions of the acellular supernatant liquid were treated in Amicon mod.8101 ultrafiltration equipment. The system consisted of a cartridge(Amicon) with a cut-off of 30,000 daltons containing 55 hollowultrafiltration fibres, a peristaltic pump and a reserve container forthe sample.

The liquid retained by the fibres returned to the reserve container ofthe ultrafiltration system whilst the liquid (containing substances withlow molecular weights) was removed from the system. The surfactin wasrecovered from the reserve container.

Upon HPLC analysis, it was observed that more than 95% of the surfactinproduced was present in the retained liquid.

b) Acidification and extraction with organic solvents

The acellular supernatant liquid and the foam from the fermentationprocess, or the liquid from step a) were brought to pH 2.0 by theaddition of 6N HCl.

The resulting flocculate was separated from the solution by centrifugingat 10,000 rpm for 15 minutes in a Sorval centrifuge (rotor Mod. GS-3)and dried under vacuum with the use of a Rotavapor 461 (Buchi,Switzerland).

The crude product thus isolated was resuspended in organic solvents suchas chloroform or dichloromethane and, after stirring for one night, theresulting mixture was filtered through Whatmann No. 4 filter paper toremove the coarser impurities.

The filtrate thus obtained was then extracted twice with equal volumesof distilled H₂ O with a pH between 7.0 and 9.0 with gentle stirring forabout 15 minutes. After this period, the mixture was placed in aseparating funnel and left for several hours to allow the two phases toseparate. The aqueous phases containing the surfactin were withdrawn,combined and acidified to pH 2.0 by the addition of 6N HCl. Thesurfactin, which was precipitated in the form of whitish granules, wasrecovered by centrifuging at 10,000 rpm, dried under vacuum and possiblyweighed to evaluate the yield.

EXAMPLE 8 Characterisation of the purified surfactin A) HPLCChromatographic analysis

Samples of purified surfactin were dissolved in an elution buffer [50%n-propanol in 0.1% TFA (trifluoracetic acid)] to give a finalconcentration of between 0.1 and 1.0 mg/ml and then analysed by HPLC.

A Beckman chromatographic system was used and was constituted by twopumps Mod. 110 B, a controller Mod. 421 A, a UV detector Mod. 163 and aShimadzu integrator mod. C-R3A.

A Licrospher® 100 RP-18 column (5 μm) with a Licrospher® 100 RP-18precolumn (5 μm) (Merck) was used for the chromatographic separations.

The chromatographic conditions were:

    ______________________________________                                        temperature    ambient (20-25° C.)                                     flow rate      0.5 ml/minute                                                  UV detector    220 nm                                                         eluent         50% n-propanol in 0.1% TFA                                     volume injected                                                                              100 μl                                                      separation time                                                                              30-40 minutes.                                                 ______________________________________                                    

The HPLC analysis was used to characterise the purity and quantity ofthe chromatographic sample and also to follow the production ofsurfactin over a period of time during the fermentation.

The standard curve shown in FIG. 14, in which the sum of the areas ofsix peaks is plotted as a function of the concentration of thesurfactin, was used for this purpose.

An equal volume of n-propanol +0.1% TFA was added to samples of theacellular supernatant liquid (50μl) possibly diluted with distilledwater, and then injected into the column.

FIG. 15 shows the separation of a 50 μg sample of purified surfactin,and FIG. 16 shows the surfactin produced during fermentation withrecycling after the strain had been grown for 3, 4, 27 and 30 hours.

B) Mass spectroscopy

The molecular weights of the compounds under test were determined bymass spectrometry.

A Finnigan mass spectrometer mod. MAT/90 equipped with an Iontech Atomgun supplied with Xenon gas accelerated at 7KV by FAB ionisation (FastAtom Bombardment) was used for the analysis.

Either glycerol-thioglycerol (1:1, v/v) or p-nitrobenzyl alcohol wasused as the matrix.

The mass spectra were recorded by a data integration system andcalibration was effected automatically with the use of aperfluorokerosine mixture (PFK).

The analysis was carried out both on purified surfactin samples and onindividual fractions produced by preparatory chromatographic separation(HPLC) carried out as described in point A).

The spectra for the former samples indicated the presence of three mainpeaks with masses [M+H]⁺ of 1008, 1022 and 1036 respectively (FIG. 17).

These results conform to those given by C.N. Mulligan et al. [Appl.Microbiol. Biotechnol., 31: 486-489, (1989)].

Mass spectra obtained for the individual fractions showed the presenceof at least 6 different components the nature of which can be explainedby the presence of fatty-acid molecules with normal, iso and anteisostructures.

C) Infrared analysis (IR)

In order to confirm the exact structure of the surfactin obtained withthe use of the mutated B.subtilis SMS 274 strain, a purified sample wassubjected to infrared analysis by the following method:

after the sample had been dispersed in KBr (1 mg of surfactin in 300 mgof KBr), the resulting suspension was ground in an agate dish in avibration mill for about 5 minutes, homogenised and then placed in atablet press. The sample was subjected to a pressure of 10 tonnes/cm² toproduce a transparent pellet which was dried at 60° C. under vacuum forone night.

The sample thus treated was then analysed by a spectrometer with aDigilab interferometer mod. FTS15E.

The data of the spectrum (FIG. 18) may be interpreted thus:

1. C-H region

The absorption bands at 2958, 2929 and 2856 cm⁻¹ are predominant andindicate the --CH₃ group. The data also show the presence of a largenumber of --CH₂ groups. The band at 1470 cm⁻¹ is a vibration deformationof the --CH² and --CH³ groups.

2. C═O region

The strong band at 1649 cm⁻¹ is due to secondary amides. The secondstrong band at 1539 cm⁻¹ corresponds to the --CO--NH--R group. A thirdsemi-weak amide band is observed at 1261 cm⁻¹, whilst a strong bandobserved at 1734 cm⁻¹ is due to a carbonyl group.

3. CH₂, CH₃ bending deltaCH₂ delta CH₃ between 1470 and 1380 cm⁻¹

1470 =deltaCH₂ +deltaCH₃

1380 delta_(sym) CH₃ is stronger and indicates a preponderance of CH₃,the presence of geminal CH₃ s and, probably, the effect of a C=O atbeta.

4. (O--H) and (N--H) Absorption

The spectrum shows vibrations of the O--H and N--H bonds at 3070 and3300 cm⁻¹. These correspond to the following groups:

3070 cm⁻¹ : --CO--NH

3300 cm⁻¹ : ═N--H

The results obtained conformed to those published by H. Kratzschmar etal [J. of Bacteriol., 170: 5347-5353, (1988)].

We claim:
 1. A method for producing surfactin comprising the stepsof:(a) growing, in a fermentation vessel, Bacillus subtilis strain ATCC55033 under aerobic conditions in a liquid culture medium containingassimilable sources of carbon, nitrogen, and inorganic salts, andoptionally containing vitamins and amino acids, at a pH o between 6.0and 7.2 and a temperature of between 25° and 42° C. wherein the amountof surfactin produced is at least 2.0 g/liter of crude surfactin in 40hours, (b) removing foam formed from the fermentation vessel, and (c)separating and purifying surfactin from the culture medium.
 2. Themethod according t claim 1, wherein the culture medium present in thefoam removed in step (b) is recycled to step (a).
 3. The methodaccording to claim 1, wherein the surfactin present in the culturemedium and in he foam is removed by an ultrafiltration system and theculture medium, less the surfactin, is recycled to step (a).
 4. Themethod according to claim 1, wherein the assimilable source of carbon isselected from the group consisting of carbohydrates, hydrolysed starchesand molasses.
 5. The method according to claim 4, wherein saidcarbohydrate is selected from the group consisting of glucose andsucrose.
 6. The method according to claim 5, wherein said carbohydrateis glucose.
 7. The method according to claim 1, wherein the assimilablesource of nitrogen is selected form eh group consisting of mineralammonium slats, urea and substances containing organic or inorganicnitrogen.
 8. The method according to claims 7, wherein said mineralammonium slats are selected from the group consisting of ammoniumnitrate, ammonium sulfate, ammonium chloride and ammonium carbonate. 9.The method according to claim 7, wherein said substances containingorganic or inorganic nitrogen are selected from the group consisting ofpeptone, yeast extract and meat extract.
 10. The method according toclaim 1, wherein said amino acids are selected from the group consistingof L-leucine, D-leucine, L-valine and L-isoleucine.
 11. The methodaccording to claim 1, wherein there is obtained at least 1.2 g/litre ofpurified surfactin in step (c).